Separation of proteins on polyacrylamide gels is one of the most popular methods used in proteomics and biochemistry research today. While considerable attention is being paid in recent years to the development of automated multi-dimensional chromatographic techniques for protein identification, a predominant amount of proteomic work is still performed using polyacrylamide gel separations. Introduction of the pre-cast two-dimensional gels and immobilized pH gradient media have considerably simplified the art of 2D protein separation. Methods based on various combinations of chromatography and one-dimensional gel separations are also becoming popular, particularly capitalizing on the fact that SDS-PAGE separation (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is, perhaps, the most efficient way to remove protein-bound detergents. Chromatographic alternatives are yet to match convenience and high resolution with respect to protein separation, offered by polyacrylamide gels in two-dimensional format as well as in a single dimension, either isoelectric focusing (IEF) or one-dimensional SDS-PAGE.
With the wide availability of high-end mass spectrometry instrumentation and growing popularity of stable isotope labeling techniques, it has become apparent that conventional in-gel digestion procedures suffer from incomplete digestion of proteins and problems related to the recovery of certain peptides out of the gel (Havli{hacek over (s)} et al., 2004). Previous reports describe many attempts to analyze the nature of the losses and eliminate some of their causes by using optimization of the methods (Terry et al., 2004; Kumarathasan et al., 2005; Castellanos-Serra et al., 2005; Bergen et al., 2005; Finehout et al., 2003; Yokono et al., 2003; Katayama et al., 2003; Lu et al., 2005), detergents (Nomura et al., 2004; Katayama et al., 2003), organic solvents (Russell et al., 2001; Havli{hacek over (s)} et al. 2003; Bunai et al., 2003; Strader et al., 2006), microwave radiation (Lopez-Ferrer et al., 2005) or ultrasound (Sun et al., 2006). However, the underlying problems still remain partially unsolved, as illustrated by one-dimensional gel electrophoresis followed by electrospray tandem mass spectrometry (GeLCMS) and SILAC isotopic labeling techniques (de Godoy et al., 2006).
During the separation in polyacrylamide gel, either in SDS-PAGE or in a second dimension of a 2D electrophoresis method, proteins coated by charged SDS molecules are driven through the pores of the polyacrylamide gel matrix by considerable forces in the electric field. Once the electric field is turned off and SDS is removed by gel fixation, proteins are thought to remain in the gel matrix during staining, imaging, spot excision, washes and de-staining. However, it is also assumed that another protein molecule, such as trypsin (ca. 24 kDa protein) or larger proteolytic enzymes, penetrate the gel matrix with no restriction during rehydration of gel plugs during the in-gel digestion step. In fact, proteases do enter the gel. However, it is expected that their concentration in the region of the highest substrate concentration, in the middle of the gel plug, would be considerably smaller than on the gel plug periphery, as a gel filtration phenomenon should take place and restrict protease entry into the gel matrix during gel plug rehydration. This phenomenon typically results in limited access of the enzyme to its substrate during proteolytic in-gel digestion, leading to diminishing peptide recovery and the occurrence of random missed cleavages. Similarly, extraction of certain products of proteolytic cleavage, particularly large hydrophobic peptides, is also restricted by the gel matrix. Furthermore, many traditional manual protocols and their robotic implementations call for organic solvent extraction steps. If the gel plugs are large enough, addition of 50% acetonitrile dehydrates the gel, beginning from the surface of the gel plug, sufficiently shrinking the acrylamide lattice to restrict peptide diffusion through it. Application of mild detergents during in-gel digestion steps has been shown to mitigate this problem to some extent by keeping the proteins and peptides in a more soluble state (Katayama et al., 2004). Nevertheless, mechanical obstruction for enzyme penetration into the gel and for peptide extraction out of it still presents a problem.
A common trend, employed by many groups to minimize this problem, is a manual cutting of the gel bands with the razor blade into cubes approximately 1 mm3 in size to improve enzymatic digestion and peptide recovery from the polyacrylamide gel matrix. However, the elevated risk of protein loss by diffusion from smaller gel pieces during washing and de-staining steps can preclude the use of this technique immediately after gel excision, while manual cutting of gel plugs after they have been washed and de-stained inevitably can lead to errors due to loss of gel plugs, which are practically invisible in their rehydrated state.
A wide variety of robotic gel processors is offered by several manufacturers. However, commercial robotic in-gel digestion systems often exceed the throughput requirements of many laboratories, frequently forcing a user to work in a 96-well format, regardless of the number of samples being processed. The growing popularity of Gel-LC-MS workflow reduces throughput requirements even further, while many large robotic systems are not optimized to handle large segments of SDS-PAGE gels containing several protein bands.